Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition

The L-type amino acid transporter 1 (LAT1) is an Na(+)-independent neutral amino acid transporter subserving the amino acid transport system L. Because of its broad substrate selectivity, system L has been proposed to be responsible for the permeation of amino acid-related drugs through the plasma membrane. To understand the mechanisms of substrate recognition, we have examined the LAT1-mediated transport using a Xenopus laevis oocyte expression system. LAT1-mediated [(14)C]phenylalanine uptake was strongly inhibited in a competitive manner by aromatic-amino acid derivatives including L-dopa, alpha-methyldopa, melphalan, triiodothyronine, and thyroxine, whereas phenylalanine methyl ester, N-methyl phenylalanine, dopamine, tyramine, carbidopa, and droxidopa did not inhibit [(14)C]phenylalanine uptake. Gabapentin, a gamma-amino acid, also exerted a competitive inhibition on LAT1-mediated [(14)C]phenylalanine uptake. Although most of the compounds that inhibited LAT1-mediated uptake were able to induce the efflux of [(14)C]phenylalanine preloaded to the oocytes expressing LAT1 through the obligatory exchange mechanism, melphalan, triiodothyronine, and thyroxine did not induce the significant efflux. Based on the experimental and semiempirical computational analyses, it is proposed that, for an aromatic amino acid to be a LAT1 substrate, it must have a free carboxyl and an amino group. The carbonyl oxygen closer to the amino group needs a computed charge of -0.55 approximately -0.56 and must not participate in hydrogen bonding. In addition, the hydrophobic interaction between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding. A substrate, however, becomes a blocker once Connolly accessible areas become large and/or the molecule has a high calculated logP value, such as those for melphalan, triiodothyronine, and thyroxine.     

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.     

Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells)

System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na⁺-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood–tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood–retinal barrier. [³H]L-leucine uptake by ARPE-19 cells took place in an Na⁺-, Cl^?-independent and saturable manner with K_m values of 8.71 and 220 µM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [³H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2475–2482, 2010     

Involvement of reduced folate carrier 1 in the inner blood-retinal barrier transport of methyltetrahydrofolate

The purpose of this study was to elucidate the mechanism of methyltetrahydrofolate (MTF) transport at the inner blood-retinal barrier (inner BRB). The characteristics and function of MTF transport at the inner BRB were examined using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. The [3H]MTF uptake by TR-iBRB2 cells increased with lowering extracellular pH and was Na+- and Cl--independent. The [3H]MTF uptake was concentration-dependent with a K(m) of 5.1 microM. This process was inhibited by reduced folate carrier 1 (RFC1) substrates, such as methotrexate and formyltetrahydrofolate, in a concentration-dependent manner with an IC50 of 8.7 and 2.8 microM, respectively, suggesting that RFC1 mediates MTF uptake in TR-iBRB2 cells. Although both RFC1 and proton-coupled folate transporter (PCFT) mRNA, which are pH-sensitive folate transporters, are expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells, the expression level of RFC1 mRNA was 83- and 49-fold greater than that of PCFT, respectively. Taken together, the above findings are consistent with the involvement of RFC1 in the inner BRB transport of MTF.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

Involvement of a Novel Organic Cation Transporter in Verapamil Transport Across the Inner Blood-Retinal Barrier

Purpose

To clarify the transport and inhibition characteristics involved in verapamil transport across the inner blood-retinal barrier (inner BRB).

Methods

The transport of [³H]verapamil across the inner BRB was investigated using retinal uptake index and integration plot analyses in rats. The detailed transport characteristics were studied using TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line that is an in vitro model of the inner BRB.

Results

The apparent influx permeability clearance of [³H]verapamil was 614 μL/(min·g retina), which is 4.7-fold greater than that of brain. The retinal uptake of [³H]verapamil was slightly increased by 3 mM verapamil and 10 mM qunidine and inhibited by 40 mM pyrilamine, supporting the carrier-mediated efflux and influx transport of verapamil across the inner BRB. TR-iBRB2 cells exhibited a concentration-dependent uptake of [³H]verapamil with a K _m of 61.9 μM, and the uptake was inhibited by several cations, such as pyrilamine, exhibiting a different profile from the identified transporters. These transport properties suggest that verapamil transport at the inner BRB takes place via a novel organic cation transporter.

Conclusions

Our findings suggest that a novel organic cation transporter is involved in verapamil transport from the blood to the retina across the inner BRB.     

L-型氨基酸转运体1 在肿瘤诊断和治疗中的研究进展

L-型氨基酸转运体1（LAT1）是L-型氨基酸转运蛋白家族的一个成员,它是由两条多肽链通过二硫键形成的异二聚体,也是异二聚体氨基酸转运体SLC7 亚家族的重要成员。LAT1 的主要功能是介导一些分子中含有苯环或者支链的、分子质量较大的、体液环境中酸碱性呈中性的氨基酸（L-亮氨酸、L-甲硫氨酸和L-苯丙氨酸等）及其类似物（美法仑、多巴和甲状腺素等）的跨膜转运,其跨膜转运方式为不依赖Na+和ATP 的协助扩散。LAT1 转运体在许多肿瘤细胞中有特异性的高表达,且其表达程度与临床上肿瘤的分期情况及治疗预后有密切的联系。LAT1 不仅被证明在肿瘤诊断中具有重要意义,也是一个潜在的肿瘤治疗靶点。本文对LAT1 的结构和转运特点进行了阐述,并总结了LAT1 在肿瘤诊断和治疗中的最新研究进展。     

Propranolol Transport Across the Inner Blood–Retinal Barrier: Potential Involvement of a Novel Organic Cation Transporter

The influx transport of propranolol across the inner blood–retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [³H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [³H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [³H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [³H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.     

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B₂) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [³H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [³H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [³H]riboflavin uptake with a K_m of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [³H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na⁺- and Cl⁻-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na⁺- and Cl⁻-independent. [³H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.     

Identification and functional characterization of a Na(+)-independent large neutral amino acid transporter (LAT2) on ARPE-19 cells

The objective of this study was to investigate the presence of a large neutral amino acid transporter on the ARPE-19 cell line. ARPE-19 cells were grown on 24-well plates for uptake studies. Uptake characteristics of [3H]L-phenylalanine (L-Phe) were determined at various concentrations and pH at 37 degrees C. Inhibition studies were conducted in presence of L- and D-amino acids, metabolic inhibitors, like ouabain, sodium azide, and in presence of sodium-free medium, to delineate the mechanism of uptake. RT-PCR was carried out on total RNA isolated from the ARPE-19 cells. Presence of Na(+)-free buffer did reduce the uptake rate. Hence, all experiments were carried out in Na(+)-free medium to delineate the sodium-independent uptake mechanism. Uptake of L-Phe on ARPE cells was found to be saturable with a Km = 89.35 +/- 14 microM, Vmax = 58.9 +/- 2.5 pmol min(-1) mg protein(-1), and Kd = 0.108 +/- 0.04 microl min(-1) mg protein(-1). Dose-dependent inhibition was observed with increasing concentrations of unlabeled L-Phe. Uptake also was found to be energy independent. Significant inhibition of [3H]L-Phe was observed with large neutral aromatic and aliphatic amino acids as well as small neutral amino acids. System L-specific inhibitor BCH produced partial inhibition of uptake. Neither acidic nor basic amino acids altered the uptake rate. Results obtained were predominantly characteristic of LAT2, particularly with respect to substrate selectivity and pH dependence. Bands for LAT2 were detected by RT-PCR in the ARPE cell line. This study provides biochemical evidence of the presence of a Na(+)-independent, facilitative transport system, LAT2, on the ARPE-19 cells.     

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [³H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [³H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [³H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [³H]3-OMG uptake was increased at 48 h. However, [³H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [³H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [³H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.     

Modulating blood-brain barrier interactions of amino acid-based anticancer agents

The large neutral amino acid (LNAA) transporter at the blood-brain barrier (BBB) mediates brain uptake of amino acid-based anticancer agents (e.g., melphalan and acivicin). In this study, we blocked the amino acid terminus of the anticancer agents using a bioreductive drug delivery system (TDDS). This molecular modification of the anticancer agents is expected to prevent LNAA carrier-mediated transport across the BBB. In this study, we demonstrate that the parent amino acid containing anticancer agents are substrates for the LNAA transporter at the BBB, whereas the TDDS is not recognized by the LNAA transporter. An in situ rat brain perfusion technique was used to determine competition for LNAA carrier-mediated transport at the BBB using [¹⁴C]L-leucine. The BBB capillary permeability-surface area (PA) product for the radiotracer [¹⁴C]L-leucine (control) was determined to be 5.18 +/- 0.32 x 10^-2 ml/s/g (100%). The control PA value for [¹⁴C]L-leucine was competitively inhibited (down to 7-18% of control) by excess L-phenylalanine as well as by excess concentration of the anticancer amino acids, melphalan and acivicin, showing competition for the LNAA transporter at the BBB. In contrast, brain perfusion of [¹⁴C]L-leucine in presence of excess TDDS resulted in no competition for brain uptake of [¹⁴C]L-leucine via the LNAA transporter. Thus, bioreversible derivatization of the parent anticancer amino acids resulted in blocking the amino acid functional group, thereby leading to loss of recognition for the cerebrovascular LNAA transporter at the BBB.     

L-type Amino Acid Transporter 1 (SLC7A5)-Mediated Transport of Pregabalin at the Rat Blood-Spinal Cord Barrier and its Sensitivity to Plasma Branched-Chain Amino Acids

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.     

Evaluation of blood-brain barrier transporters by co-culture of brain capillary endothelial cells with astrocytes

To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as gamma-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [(3)H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [(14)C]benzoic acid was saturable, and the K(m) value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [(14)C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [(3)H]dehydroepiandrosterone sulfate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.